Novel human kinase and polynucleotides encoding the same

ABSTRACT

Novel human polynucleotide and polypeptide sequences are disclosed that can be used in therapeutic, diagnostic, and pharmacogenomic applications.

[0001] The present application claims the benefit of U.S. ProvisionalApplication No. 60/277,168 which was filed on Mar. 20, 2001 and isherein incorporated by reference in its entirety.

1. INTRODUCTION

[0002] The present invention relates to the discovery, identification,and characterization of novel human polynucleotides encoding a kinasewhich share sequence similarity with animal kinases. The inventionencompasses the described polynucleotides, host cell expression systems,the encoded proteins, fusion proteins, polypeptides and peptides,antibodies to the encoded proteins and peptides, and geneticallyengineered animals that either lack or overexpress the disclosed genes,antagonists and agonists of the proteins, and other compounds thatmodulate the expression or activity of the proteins encoded by thedisclosed genes, which can be used for diagnosis, drug screening,clinical trial monitoring, the treatment of diseases and disorders, andcosmetic or nutriceutical applications.

2. BACKGROUND OF THE INVENTION

[0003] Kinases mediate the phosphorylation of a wide variety of proteinsand compounds in the cell. In conjunction with phosphatases, kinases areinvolved in a range of regulatory pathways. Given the physiologicalimportance of kinases, they have been subject to intense scrutiny andare proven drug targets.

3. SUMMARY OF THE INVENTION

[0004] The present invention relates to the discovery, identification,and characterization of nucleotides that encode novel a human kinase andthe corresponding amino acid sequence of this protein. The novel humankinases (NHK) described for the first time herein shares structuralsimilarity with animal kinases, including, but not limited to, receptortyrosine kinases and especially kinases of the membrane-associatedguanylate kinase (MAGUK) family. The described NHK encoding sequencesdefine a novel kinase having homologues and orthologs across a range ofphyla and species.

[0005] The novel human polynucleotide described herein, encode an openreading frame (ORF) of 455 amino acids in length (see SEQ ID NO: 2).

[0006] The invention also encompasses agonists and antagonists of thedescribed NHK, including small molecules, large molecules, mutant NHKs,or portions thereof, that compete with native NHK, peptides, andantibodies, as well as nucleotide sequences that can be used to inhibitthe expression of the described NHK (e.g., antisense and ribozymemolecules, and open reading frame or regulatory sequence replacementconstructs) or to enhance the expression of the described NHK (e.g.,expression constructs that place the described polynucleotide under thecontrol of a strong promoter system), and transgenic animals thatexpress a NHK sequence, or “knock-outs” (which can be conditional) thatdo not express a functional NHK. Such knock-out mice can be produced inseveral ways, one of which involves the use of mouse embryonic stem cell(“ES cells”) lines that contain gene trap mutations in a murine homologthe described NHK. When the unique NHK sequences described in SEQ IDNOS:1-2 are “knocked-out” they provide a method of identifyingphenotypic expression of the particular gene as well as a method ofassigning function to previously unknown genes. In addition, animals inwhich the unique NHK sequences described in SEQ ID NOS:1-2 are“knocked-out” provide a unique source in which to elicit antibodies tohomologous and orthologous proteins which would have been previouslyviewed by the immune system as “self” and therefore would have failed toelicit significant antibody responses.

[0007] Additionally, the unique NHK sequences described in SEQ IDNOS:1-2 are useful for the identification of protein coding sequence andmapping a unique gene to a particular chromosome. These sequencesidentify actual, biologically verified, and therefore relevant, exonsplice junctions as opposed to those that may have beenbioinformatically predicted from genomic sequence alone. The sequencesof the present invention are also useful as additional DNA markers forrestriction fragment length polymorphism (RFLP) analysis, and inforensic biology.

[0008] Further, the present invention also relates to processes foridentifying compounds that modulate, i.e., act as agonists orantagonists, of NHK expression and/or NHK activity that utilize purifiedpreparations of the described NHK and/or NHK products, or cellsexpressing the same. Such compounds can be used as therapeutic agentsfor the treatment of any of a wide variety of symptoms associated withbiological disorders or imbalances.

4. DESCRIPTION OF THE SEQUENCE LISTING AND FIGURES

[0009] The Sequence Listing provides the sequence of the novel human ORFencoding the described novel human kinase protein.

5. DETAILED DESCRIPTION OF THE INVENTION

[0010] The NHK described for the first time herein defines a novelprotein that is expressed in, inter alia, human cell lines and fetalbrain, spinal cord, lymph node, bone marrow, adrenal gland, fetalkidney, and fetal lung cells.

[0011] The present invention encompasses the nucleotides presented inthe Sequence Listing, host cells expressing such nucleotides, theexpression products of such nucleotides, and: (a) nucleotides thatencode mammalian homologs of the described genes, including thespecifically described NHK, and related NHK products; (b) nucleotidesthat encode one or more portions of a NHK that correspond to functionaldomains, and the polypeptide products specified by such nucleotidesequences including, but not limited to, the novel regions of any activedomain(s); (c) isolated nucleotides that encode mutant versions,engineered or naturally occurring, of the described NHK in which all ora part of at least one domain is deleted or altered, and the polypeptideproducts specified by such nucleotide sequences including, but notlimited to, soluble proteins and peptides in which all or a portion ofthe signal sequence is deleted; (d) nucleotides that encode chimericfusion proteins containing all or a portion of a coding region of a NHK,or one of its domains (e.g., a receptor/ligand binding domain, accessoryprotein/self-association domain, etc.) fused to another peptide orpolypeptide; or (e) therapeutic or diagnostic derivatives of thedescribed polynucleotides such as oligonucleotides, antisensepolynucleotides, ribozymes, dsRNA, or gene therapy constructs comprisinga sequence first disclosed in the Sequence Listing. As discussed above,the present invention includes: (a) the human DNA sequences presented inthe Sequence Listing (and vectors comprising the same) and additionallycontemplates any nucleotide sequence encoding a contiguous NHK openreading frame (ORF) that hybridizes to a complement of a DNA sequencepresented in the Sequence Listing under highly stringent conditions,e.g., hybridization to filter-bound DNA in 0.5 M NaHPO₄, 7% sodiumdodecyl sulfate (SDS), 1 mM EDTA at 65° C., and washing in 0.1×SSC/0.1%SDS at 68° C. ((Ausubel F. M. et al., eds., 1989, Current Protocols inMolecular Biology, Vol. I, Green Publishing Associates, Inc., and JohnWiley & Sons, Inc., NY, at p. 2.10.3) and encodes a functionallyequivalent expression product.

[0012] Additionally contemplated are any nucleotide sequences thathybridize to the complement of the DNA sequence that encode and expressan amino acid sequence presented in the Sequence Listing undermoderately stringent conditions, e.g., washing in 0.2×SSC/0.1% SDS at42° C. (Ausubel et al., 1989, supra), yet still encode a functionallyequivalent NHK product. Functional equivalents of a NHK includenaturally occurring NHKs present in other species and mutant NHKswhether naturally occurring or engineered (by site directed mutagenesis,gene shuffling, directed evolution as described in, for example, U.S.Pat. No. 5,837,458 or 5,723,323 both of which are herein incorporated byreference). The invention also includes degenerate nucleic acid variantsof the disclosed NHK polynucleotide sequences.

[0013] Additionally contemplated are polynucleotides encoding NHK ORFs,or their functional equivalents, encoded by polynucleotide sequencesthat are about 99, 95, 90, or about 85 percent similar to correspondingregions of a NHK (as measured by BLAST sequence comparison analysisusing, for example, the GCG sequence analysis package using defaultparameters).

[0014] The invention also includes nucleic acid molecules, preferablyDNA molecules, that hybridize to, and are therefore the complements of,the described NHK encoding polynucleotides. Such hybridizationconditions can be highly stringent or less highly stringent, asdescribed above. In instances where the nucleic acid molecules aredeoxyoligonucleotides (“DNA oligos”), such molecules are generally about16 to about 100 bases long, or about 20 to about 80, or about 34 toabout 45 bases long, or any variation or combination of sizesrepresented therein that incorporate a contiguous region of sequencefirst disclosed in the Sequence Listing. Such oligonucleotides can beused in conjunction with the polymerase chain reaction (PCR) to screenlibraries, isolate clones, and prepare cloning and sequencing templates,etc.

[0015] Alternatively, such NHK oligonucleotides can be used ashybridization probes for screening libraries, and assessing geneexpression patterns (particularly using a micro array or high-throughput“chip” format). Additionally, a series of the described NHKoligonucleotide sequences, or the complements thereof, can be used torepresent all or a portion of the described NHK sequences. Anoligonucleotide or polynucleotide sequence first disclosed in at least aportion of SEQ ID NO: 1 can be used as a hybridization probe inconjunction with a solid support matrix/substrate (resins, beads,membranes, plastics, polymers, metal or metallized substrates,crystalline or polycrystalline substrates, etc.). Of particular note arespatially addressable arrays (i.e., gene chips, microtiter plates, etc.)of oligonucleotides and polynucleotides, or corresponding oligopeptidesand polypeptides, wherein at least one of the biopolymers present on thespatially addressable array comprises an oligonucleotide orpolynucleotide sequence which is first disclosed in at least a portionof SEQ ID NO: 1, or, where protein chips are contemplated, an amino acidsequence encoded thereby. Methods for attaching biopolymers to, orsynthesizing biopolymers on, solid support matrices, and conductingbinding studies thereon are disclosed in, inter alia, U.S. Pat. Nos.5,700,637, 5,556,752, 5,744,305, 4,631,211, 5,445,934, 5,252,743,4,713,326, 5,424,186, and 4,689,405 the disclosures of which are hereinincorporated by reference in their entirety.

[0016] Addressable arrays comprising sequences first disclosed in atleast a portion of SEQ ID NOS:1-2 can be used to identify andcharacterize the temporal and tissue specific expression of a gene.These addressable arrays incorporate oligonucleotide sequences ofsufficient length to confer the required specificity, yet be within thelimitations of the production technology. The length of these probes iswithin a range of between about 8 to about 2000 nucleotides. Preferablythe probes consist of 60 nucleotides and more preferably 25 nucleotidesfrom a sequence first disclosed in at least a portion of SEQ ID NO:1.

[0017] For example, a series of the described oligonucleotide sequences,or the complements thereof, can be used in chip format to represent allor a portion of the described sequences. The oligonucleotides, typicallybetween about 16 to about 40 (or any whole number within the statedrange) nucleotides in length can partially overlap each other and/or thesequence may be represented using oligonucleotides that do not overlap.Accordingly, the described polynucleotide sequences shall typicallycomprise at least about two or three distinct oligonucleotide sequencesof at least about 8 nucleotides in length that are each first disclosedin the described Sequence Listing. Such oligonucleotide sequences canbegin at any nucleotide present within a sequence in the SequenceListing and proceed in either a sense (5′-to-3′) orientation vis-a-visthe described sequence or in an antisense orientation.

[0018] Microarray-based analysis allows the discovery of broad patternsof genetic activity, providing new understanding of gene functions andgenerating novel and unexpected insight into transcriptional processesand biological mechanisms. The use of addressable arrays comprisingsequences first disclosed in at least a portion of SEQ ID NO:1 providesdetailed information about transcriptional changes involved in aspecific pathway, potentially leading to the identification of novelcomponents or gene functions that manifest themselves as novelphenotypes.

[0019] Probes consisting of sequences first disclosed in at least aportion of SEQ ID NO:1 can also be used in the identification, selectionand validation of novel molecular targets for drug discovery. The use ofthese unique sequences permits the direct confirmation of drug targetsand recognition of drug dependent changes in gene expression that aremodulated through pathways distinct from the drug's intended target.These unique sequences therefore also have utility in defining andmonitoring both drug action and toxicity.

[0020] As an example of utility, the sequences first disclosed in atleast a portion of SEQ ID NOS:1-2 can be utilized in microarrays orother assay formats, to screen collections of genetic material frompatients who have a particular medical condition. These investigationscan also be carried out using the sequences first disclosed in at leasta portion of SEQ ID NOS:1-2 in silico and by comparing previouslycollected genetic databases and the disclosed sequences using computersoftware known to those in the art.

[0021] Thus the sequences first disclosed in at least a portion of SEQID NOS:1-2 can be used to identify mutations associated with aparticular disease and also as a diagnostic or prognostic assay.

[0022] Although the presently described sequences have been specificallydescribed using nucleotide sequence, it should be appreciated that eachof the sequences can uniquely be described using any of a wide varietyof additional structural attributes, or combinations thereof. Forexample, a given sequence can be described by the net composition of thenucleotides present within a given region of the sequence in conjunctionwith the presence of one or more specific oligonucleotide sequence(s)first disclosed in at least a portion of SEQ ID NO: 1. Alternatively, arestriction map specifying the relative positions of restrictionendonuclease digestion sites, or various palindromic or other specificoligonucleotide sequences can be used to structurally describe a givensequence. Such restriction maps, which are typically generated by widelyavailable computer programs (e.g., the University of Wisconsin GCGsequence analysis package, SEQUENCHER 3.0, Gene Codes Corp., Ann Arbor,Mich., etc.), can optionally be used in conjunction with one or morediscrete nucleotide sequence(s) present in the sequence that can bedescribed by the relative position of the sequence relative to one ormore additional sequence(s) or one or more restriction sites present inthe disclosed sequence.

[0023] For oligonucleotide probes, highly stringent conditions mayrefer, e.g., to washing in 6×SSC/0.05% sodium pyrophosphate at 37° C.(for 14-base oligos), 48° C. (for 17-base oligos), 55° C. (for 20-baseoligos), and 60° C. (for 23-base oligos). These nucleic acid moleculesmay encode or act as NHK gene antisense molecules, useful, for example,in NHK gene regulation and/or as antisense primers in amplificationreactions of NHK gene nucleic acid sequences. With respect to NHK generegulation, such techniques can be used to regulate biologicalfunctions. Further, such sequences can be used as part of ribozymeand/or triple helix sequences that are also useful for NHK generegulation.

[0024] Inhibitory antisense or double stranded oligonucleotides canadditionally comprise at least one modified base moiety which isselected from the group including, but not limited to, 5-fluorouracil,5-bromouracil, 5-chlorouracil, 5-iodouracil, hypoxanthine, xanthine,4-acetylcytosine, 5-(carboxyhydroxylmethyl) uracil,5-carboxymethylaminomethyl-2-thiouridine,5-carboxymethylaminomethyluracil, dihydrouracil,beta-D-galactosylqueosine, inosine, N6-isopentenyladenine,1-methylguanine, 1-methylinosine, 2,2-dimethylguanine, 2-methyladenine,2-methylguanine, 3-methylcytosine, 5-methylcytosine, N6-adenine,7-methylguanine, 5-methylaminomethyluracil,5-methoxyaminomethyl-2-thiouracil, beta-D-mannosylqueosine,5′-methoxycarboxymethyluracil, 5-methoxyuracil,2-methylthio-N-6-isopentenyladenine, uracil-5-oxyacetic acid (v),wybutoxosine, pseudouracil, queosine, 2-thiocytosine,5-methyl-2-thiouracil, 2-thiouracil, 4-thiouracil, 5-methyluracil,uracil-5-oxyacetic acid methylester, uracil-5-oxyacetic acid (v),5-methyl-2-thiouracil, 3-(3-amino-3-N-2-carboxypropyl) uracil, (acp3)w,and 2,6-diaminopurine.

[0025] The antisense oligonucleotide can also comprise at least onemodified sugar moiety selected from the group including, but not limitedto, arabinose, 2-fluoroarabinose, xylulose, and hexose.

[0026] In yet another embodiment, the antisense oligonucleotide willcomprise at least one modified phosphate backbone selected from thegroup including, but not limited to, a phosphorothioate, aphosphorodithioate, a phosphoramidothioate, a phosphoramidate, aphosphordiamidate, a methylphosphonate, an alkyl phosphotriester, and aformacetal or analog thereof.

[0027] In yet another embodiment, the antisense oligonucleotide is anα-anomeric oligonucleotide. An α-anomeric oligonucleotide forms specificdouble-stranded hybrids with complementary RNA in which, contrary to theusual β-units, the strands run parallel to each other (Gautier et al.,1987, Nucl. Acids Res. 15:6625-6641). The oligonucleotide is a2′-0-methylribonucleotide (Inoue et al., 1987, Nucl. Acids Res.15:6131-6148), or a chimeric RNA-DNA analogue (Inoue et al., 1987, FEBSLett. 215:327-330). Alternatively, double stranded RNA can be used todisrupt the expression and function of a targeted NHK.

[0028] Oligonucleotides of the invention can be synthesized by standardmethods known in the art, e.g. by use of an automated DNA synthesizer(such as are commercially available from Biosearch, Applied Biosystems,etc.). As examples, phosphorothioate oligonucleotides can be synthesizedby the method of Stein et al. (1988, Nucl. Acids Res. 16:3209), andmethylphosphonate oligonucleotides can be prepared by use of controlledpore glass polymer supports (Sarin et al., 1988, Proc. Natl. Acad. Sci.U.S.A. 85:7448-7451), etc.

[0029] Low stringency conditions are well-known to those of skill in theart, and will vary predictably depending on the specific organisms fromwhich the library and the labeled sequences are derived. For guidanceregarding such conditions see, for example, Sambrook et al., 1989,Molecular Cloning, A Laboratory Manual (and periodic updates thereof),Cold Spring Harbor Press, NY; and Ausubel et al., 1989, CurrentProtocols in Molecular Biology, Green Publishing Associates and WileyInterscience, NY.

[0030] Alternatively, suitably labeled NHK nucleotide probes can be usedto screen a human genomic library using appropriately stringentconditions or by PCR. The identification and characterization of humangenomic clones is helpful for identifying polymorphisms (including, butnot limited to, nucleotide repeats, microsatellite alleles, singlenucleotide polymorphisms, or coding single nucleotide polymorphisms),determining the genomic structure of a given locus/allele, and designingdiagnostic tests. For example, sequences derived from regions adjacentto the intron/exon boundaries of the human gene can be used to designprimers for use in amplification assays to detect mutations within theexons, introns, splice sites (e.g., splice acceptor and/or donor sites),etc., that can be used in diagnostics and pharmacogenomics.

[0031] In another example, the present sequences can be used inrestriction fragment length polymorphism (RFLP) analysis to identifyspecific individuals. In this technique, an individual's genomic DNA isdigested with one or more restriction enzymes, and probed on a Southernblot to yield unique bands for identification (as generally described inU.S. Pat. No. 5,272,057, incorporated herein by reference). In addition,the sequences of the present invention can be used to providepolynucleotide reagents, e.g., PCR primers, targeted to specific loci inthe human genome, which can enhance the reliability of DNA-basedforensic identifications by, for example, providing another“identification marker” (i.e., another DNA sequence that is unique to aparticular individual). Actual base sequence information can be used foridentification as an accurate alternative to patterns formed byrestriction enzyme generated fragments.

[0032] Further, a NHK gene homolog can be isolated from nucleic acidfrom an organism of interest by performing PCR using two degenerate or“wobble” oligonucleotide primer pools designed on the basis of aminoacid sequences within the NHK products disclosed herein. The templatefor the reaction may be total RNA, mRNA, and/or cDNA obtained by reversetranscription of mRNA prepared from, for example, human or non-humancell lines or tissue known or suspected to express an allele of the NHKencoding genomic locus/gene.

[0033] The PCR product can be subcloned and sequenced to ensure that theamplified sequences represent the sequence of the desired NHK gene. ThePCR fragment can then be used to isolate a full length cDNA clone by avariety of methods. For example, the amplified fragment can be labeledand used to screen a cDNA library, such as a bacteriophage cDNA library.Alternatively, the labeled fragment can be used to isolate genomicclones via the screening of a genomic library.

[0034] PCR technology can also be used to isolate full length cDNAsequences. For example, RNA can be isolated, following standardprocedures, from an appropriate cellular or tissue source (i.e., oneknown, or suspected, to express a NHK gene). A reverse transcription(RT) reaction can be performed on the RNA using an oligonucleotideprimer specific for the most 5′ end of the amplified fragment for thepriming of first strand synthesis. The resulting RNA/DNA hybrid may thenbe “tailed” using a standard terminal transferase reaction, the hybridmay be digested with RNase H, and second strand synthesis may then beprimed with a complementary primer. Thus, cDNA sequences upstream of theamplified fragment can be isolated. For a review of cloning strategiesthat can be used, see e.g., Sambrook et al., 1989, supra.

[0035] A cDNA encoding a mutant NHK sequence can be isolated, forexample, by using PCR. In this case, the first cDNA strand may besynthesized by hybridizing an oligo-dT oligonucleotide to mRNA isolatedfrom tissue known or suspected to be expressed in an individualputatively carrying a mutant NHK allele, and by extending the new strandwith reverse transcriptase. The second strand of the cDNA is thensynthesized using an oligonucleotide that hybridizes specifically to the5′ end of the normal sequence. Using these two primers, the product isthen amplified via PCR, optionally cloned into a suitable vector, andsubjected to DNA sequence analysis through methods well-known to thoseof skill in the art. By comparing the DNA sequence of the mutant NHKallele to that of a corresponding normal NHK allele, the mutation(s)responsible for the loss or alteration of function of the mutant NHKgene product can be ascertained.

[0036] Alternatively, a genomic library can be constructed using DNAobtained from an individual suspected of or known to carry a mutant NHKallele (e.g., a person manifesting a NHK-associated phenotype such as,for example, immune disorders, obesity, high blood pressure, etc.), or acDNA library can be constructed using RNA from a tissue known, orsuspected, to express a mutant NHK allele. A normal NHK gene, or anysuitable fragment thereof, can then be labeled and used as a probe toidentify the corresponding mutant NHK allele in such libraries. Clonescontaining mutant NHK sequences can then be purified and subjected tosequence analysis according to methods well-known to those skilled inthe art.

[0037] Additionally, an expression library can be constructed utilizingcDNA synthesized from, for example, RNA isolated from a tissue known, orsuspected, to express a mutant NHK allele in an individual suspected ofor known to carry such a mutant allele. In this manner, gene productsmade by the putatively mutant tissue may be expressed and screened usingstandard antibody screening techniques in conjunction with antibodiesraised against a normal NHK product, as described below. (For screeningtechniques, see, for example, Harlow, E. and Lane, eds., 1988,“Antibodies: A Laboratory Manual”, Cold Spring Harbor Press, Cold SpringHarbor, N.Y.)

[0038] Additionally, screening can be accomplished by screening withlabeled NHK fusion proteins, such as, for example, alkalinephosphatase-NHK or NHK-alkaline phosphatase fusion proteins. In caseswhere a NHK mutation results in an expression product with alteredfunction (e.g., as a result of a missense or a frameshift mutation),polyclonal antibodies to NHK are likely to cross-react with acorresponding mutant NHK expression product. Library clones detected viatheir reaction with such labeled antibodies can be purified andsubjected to sequence analysis according to methods well-known in theart.

[0039] An additional application of the described novel humanpolynucleotide sequences is their use in the molecularmutagenesis/evolution of proteins that are at least partially encoded bythe described novel sequences using, for example, polynucleotideshuffling or related methodologies. Such approaches are described inU.S. Pat. Nos. 5,830,721, 5,837,458, 6,117,679, and 5,723,323 which areherein incorporated by reference in their entirety.

[0040] The invention also encompasses (a) DNA vectors that contain anyof the foregoing NHK coding sequences and/or their complements (i.e.,antisense); (b) DNA expression vectors that contain any of the foregoingNHK coding sequences operatively associated with a regulatory elementthat directs the expression of the coding sequences (for example,baculovirus as described in U.S. Pat. No. 5,869,336 herein incorporatedby reference); (c) genetically engineered host cells that contain any ofthe foregoing NHK coding sequences operatively associated with aregulatory element that directs the expression of the coding sequencesin the host cell; and (d) genetically engineered host cells that expressan endogenous NHK sequence under the control of an exogenouslyintroduced regulatory element (i.e., gene activation). As used herein,regulatory elements include, but are not limited to, inducible andnon-inducible promoters, enhancers, operators and other elements knownto those skilled in the art that drive and regulate expression. Suchregulatory elements include, but are not limited to, the cytomegalovirus(hCMV) immediate early gene, regulatable, viral elements (particularlyretroviral LTR promoters), the early or late promoters of SV40adenovirus, the lac system, the trp system, the TAC system, the TRCsystem, the major operator and promoter regions of phage lambda, thecontrol regions of fd coat protein, the promoter for 3-phosphoglyceratekinase (PGK), the promoters of acid phosphatase, and the promoters ofthe yeast α-mating factors.

[0041] Where, as in the present instance, some of the described NHKpeptides or polypeptides are thought to be cytoplasmic or nuclearproteins (although processed forms or fragments can be secreted ormembrane associated), expression systems can be engineered that producesoluble derivatives of a NHK (corresponding to a NHK extracellularand/or intracellular domains, or truncated polypeptides lacking one ormore hydrophobic domains) and/or NHK fusion protein products (especiallyNHK-Ig fusion proteins, i.e., fusions of a NHK domain to an IgFc), NHKantibodies, and anti-idiotypic antibodies (including Fab fragments) thatcan be used in therapeutic applications. Preferably, the aboveexpression systems are engineered to allow the desired peptide orpolypeptide to be recovered from the culture media.

[0042] The present invention also encompasses antibodies andanti-idiotypic antibodies (including Fab fragments), antagonists andagonists of a NHK, as well as compounds or nucleotide constructs thatinhibit expression of a NHK sequence (transcription factor inhibitors,antisense and ribozyme molecules, or open reading frame sequence orregulatory sequence replacement constructs), or promote the expressionof a NHK (e.g., expression constructs in which NHK coding sequences areoperatively associated with expression control elements such aspromoters, promoter/enhancers, etc.).

[0043] The NHK or NHK peptides, NHK fusion proteins, NHK nucleotidesequences, antibodies, antagonists and agonists can be useful for thedetection of mutant NHKs or inappropriately expressed forms of NHK forthe diagnosis of disease. The NHK proteins or peptides, NHK fusionproteins, NHK nucleotide sequences, host cell expression systems,antibodies, antagonists, agonists and genetically engineered cells andanimals can be used for screening for drugs (or high throughputscreening of combinatorial libraries) effective in the treatment of thesymptomatic or phenotypic manifestations of perturbing the normalfunction of a NHK in the body. The use of engineered host cells and/oranimals can offer an advantage in that such systems allow not only forthe identification of compounds that bind to the endogenousreceptor/ligand of a NHK, but can also identify compounds that triggerNHK-mediated activities or pathways.

[0044] Finally, the NHK products can be used as therapeutics. Forexample, soluble derivatives such as NHK peptides/domains correspondingto the NHK, NHK fusion protein products (especially NHK-Ig fusionproteins, i.e., fusions of a NHK, or a domain of a NHK, to an IgFc), NHKantibodies and anti-idiotypic antibodies (including Fab fragments),antagonists or agonists (including compounds that modulate or act ondownstream targets in a NHK-mediated pathway) can be used to directlytreat diseases or disorders. For instance, the administration of aneffective amount of soluble NHK, or a NHK-IgFc fusion protein or ananti-idiotypic antibody (or its Fab) that mimics the NHK could activateor effectively antagonize the endogenous NHK or a protein interactivetherewith. Nucleotide constructs encoding such NHK products can be usedto genetically engineer host cells to express such products in vivo;these genetically engineered cells function as “bioreactors” in the bodydelivering a continuous supply of a NHK, a NHK peptide, or a NHK fusionprotein to the body. Nucleotide constructs encoding functional NHKs,mutant NHKs, as well as antisense and ribozyme molecules can also beused in “gene therapy” approaches for the modulation of NHK expression.Thus, the invention also encompasses pharmaceutical formulations andmethods for treating biological disorders.

[0045] Various aspects of the invention are described in greater detailin the subsections below.

5.1 The NHK Sequences

[0046] The cDNA sequence and corresponding deduced amino acid sequenceof the described NHK are presented in the Sequence Listing.

[0047] Expression analysis has provided evidence that the described NHKcan be expressed in a range of human tissues. The described NHKrepresents a novel MAGUK (SEQ ID NOS: 1-2). Given the physiologicalimportance of protein kinases, they have been subject to intensescrutiny as exemplified and discussed in U.S. Pat. No. 5,817,479, hereinincorporated by reference in its entirety, which describes uses andutilities that are applicable to the described NHK.

[0048] The described sequences were compiled from sequences available inGENBANK and cDNAs generated from fetal kidney, spleen, and spinal cordmRNAs (Edge Biosystems, Gaithersburg, Md.).

[0049] A G/C polymorphism was detected in SEQ ID NO:1 at position 427which can result in either an asp or his being present at correspondingamino acid position 144 of SEQ ID NO:2.

[0050] The gene encoding SEQ ID NO:1-2 is apparently present on humanchromosome 2 (see GENBANK accession no. AC067825). Accordingly, thedescribed sequences are also useful for mapping the corresponding codingregions of the human genome.

[0051] The described novel human polynucleotide sequences can be used,among other things, in the molecular mutagenesis/evolution of proteinsthat are at least partially encoded by the described novel sequencesusing, for example, polynucleotide shuffling or related methodologies.Such approaches are described in U.S. Pat. Nos. 5,830,721 and 5,837,458which are herein incorporated by reference in their entirety.

[0052] NHK gene products can also be expressed in transgenic animals.Animals of any species including, but not limited to, worms, mice, rats,rabbits, guinea pigs, pigs, micro-pigs, birds, goats, and non-humanprimates, e.g., baboons, monkeys, and chimpanzees may be used togenerate NHK transgenic animals.

[0053] Any technique known in the art may be used to introduce agenetically engineered NHK transgene into animals to produce the founderlines of transgenic animals. Such techniques include, but are notlimited to pronuclear microinjection (Hoppe, P. C. and Wagner, T. E.,1989, U.S. Pat. No. 4,873,191); retrovirus-mediated gene transfer intogerm lines (Van der Putten et al., 1985, Proc. Natl. Acad. Sci. USA82:6148-6152); gene targeting in embryonic stem cells (Thompson et al.,1989, Cell 56:313-321); electroporation of embryos (Lo, 1983, Mol Cell.Biol. 3:1803-1814); and sperm-mediated gene transfer (Lavitrano et al.,1989, Cell 57:717-723); etc. For a review of such techniques, seeGordon, 1989, Transgenic Animals, Intl. Rev. Cytol. 115:171-229, whichis incorporated by reference herein in its entirety.

[0054] The present invention provides for transgenic animals that carrythe NHK transgene in all their cells, as well as animals which carry thetransgene in some, but not all their cells, i.e., mosaic animals orsomatic cell transgenic animals. The transgene may be integrated as asingle transgene or in concatamers, e.g., head-to-head tandems orhead-to-tail tandems. The transgene may also be selectively introducedinto and activated in a particular cell-type by following, for example,the teaching of Lasko et al., 1992, Proc. Natl. Acad. Sci. USA89:6232-6236. The regulatory sequences required for such a cell-typespecific activation will depend upon the particular cell-type ofinterest, and will be apparent to those of skill in the art.

[0055] When it is desired that a NHK transgene be integrated into thechromosomal site of the endogenous NHK gene, gene targeting ispreferred. Briefly, when such a technique is to be utilized, vectorscontaining some nucleotide sequences homologous to the endogenous NHKgene are designed for the purpose of integrating, via homologousrecombination with chromosomal sequences, into and disrupting thefunction of the nucleotide sequence of the endogenous NHK gene (i.e.,“knockout” animals).

[0056] The transgene can also be selectively introduced into aparticular cell-type, thus inactivating the endogenous NHK gene in onlythat cell-type, by following, for example, the teaching of Gu et al.,1994, Science, 265:103-106. The regulatory sequences required for such acell-type specific inactivation will depend upon the particularcell-type of interest, and will be apparent to those of skill in theart.

[0057] Once transgenic animals have been generated, the expression ofthe recombinant NHK gene may be assayed utilizing standard techniques.Initial screening may be accomplished by Southern blot analysis or PCRtechniques to analyze animal tissues to assay whether integration of thetransgene has taken place. The level of mRNA expression of the transgenein the tissues of the transgenic animals may also be assessed usingtechniques which include but are not limited to Northern blot analysisof tissue samples obtained from the animal, in situ hybridizationanalysis, and RT-PCR. Samples of NHK gene-expressing tissue, may also beevaluated immunocytochemically using antibodies specific for the NHKtransgene product.

[0058] The present invention also provides for “knock-in” animals.Knock-in animals are those in which a polynucleotide sequence (i.e., agene or a cDNA) that the animal does not naturally have in its genome isinserted in such a way that the sequence is expressed. Examples include,but are not limited to, a human gene or cDNA used to replace its murineortholog in the mouse, a murine cDNA used to replace the murine gene inthe mouse, and a human gene or cDNA or murine cDNA that is tagged with areporter construct used to replace the murine ortholog or gene in themouse. Such replacements can occur at the locus of the murine orthologor gene, or at another specific site. Such knock-in animals are usefulfor the in vivo study, testing and validation of, intra alia, human drugtargets, as well as for compounds that are directed at the same andtherapeutic proteins.

5.2 NHK and NHK Polypeptides

[0059] NHK products, polypeptides, peptide fragments, mutated,truncated, or deleted forms of the NHK, and/or NHK fusion proteins canbe prepared for a variety of uses. These uses include, but are notlimited to, the generation of antibodies, as reagents in diagnosticassays, the identification of other cellular gene products related tothe NHK, as reagents in assays for screening for compounds that can beused as pharmaceutical reagents useful in the therapeutic treatment ofmental, biological, or medical disorders and disease.

[0060] The Sequence Listing discloses the amino acid sequence encoded bythe described NHK-encoding polynucleotides. The NHK displays aninitiator methionine that is present in a DNA sequence contextconsistent with eucaryotic translation initiation sites. SEQ ID NO:2displays a consensus signal sequence similar to that typically seen insecreted or membrane proteins (the MAGUKs such as the described NHK aretypically membrane proteins).

[0061] The NHK amino acid sequence of the invention includes the aminoacid sequence presented in the Sequence Listing as well as analogues andderivatives thereof. Further, corresponding NHK homologues from otherspecies are encompassed by the invention. In fact, any NHK proteinencoded by the NHK nucleotide sequences described above are within thescope of the invention, as are any novel polynucleotide sequencesencoding all or any novel portion of an amino acid sequence presented inthe Sequence Listing. The degenerate nature of the genetic code iswell-known, and, accordingly, each amino acid presented in the SequenceListing, is generically representative of the well-known nucleic acid“triplet” codon, or in many cases codons, that can encode the aminoacid. As such, as contemplated herein, the amino acid sequencespresented in the Sequence Listing, when taken together with the geneticcode (see, for example, Table 4-1 at page 109 of “Molecular CellBiology”, 1986, J. Darnell et al. eds., Scientific American Books, NewYork, N.Y., herein incorporated by reference) are genericallyrepresentative of all the various permutations and combinations ofnucleic acid sequences that can encode such amino acid sequences.

[0062] The invention also encompasses proteins that are functionallyequivalent to a NHK encoded by the presently described nucleotidesequences as judged by any of a number of criteria, including, but notlimited to, the ability to bind and modify a NHK substrate, or theability to effect an identical or complementary downstream pathway, or achange in cellular metabolism (e.g., proteolytic activity, ion flux,tyrosine phosphorylation, etc.). Such functionally equivalent NHKproteins include, but are not limited to, additions or, substitutions ofamino acid residues within the amino acid sequence encoded by the NHKnucleotide sequences described above, but which result in a silentchange, thus producing a functionally equivalent expression product.Amino acid substitutions may be made on the basis of similarity inpolarity, charge, solubility, hydrophobicity, hydrophilicity, and/or theamphipathic nature of the residues involved. For example, nonpolar(hydrophobic) amino acids include alanine, leucine, isoleucine, valine,proline, phenylalanine, tryptophan, and methionine; polar neutral aminoacids include glycine, serine, threonine, cysteine, tyrosine,asparagine, and glutamine; positively charged (basic) amino acidsinclude arginine, lysine, and histidine; and negatively charged (acidic)amino acids include aspartic acid and glutamic acid.

[0063] A variety of host-expression vector systems can be used toexpress the NHK nucleotide sequences of the invention. Where the NHKpeptide or polypeptide can exist, or has been engineered to exist, as asoluble or secreted molecule, the soluble NHK peptide or polypeptide canbe recovered from the culture media. Such expression systems alsoencompass engineered host cells that express a NHK, or functionalequivalent, in situ. Purification or enrichment of a NHK from suchexpression systems can be accomplished using appropriate detergents andlipid micelles and methods well-known to those skilled in the art.However, such engineered host cells themselves may be used in situationswhere it is important not only to retain the structural and functionalcharacteristics of the NHK, but to assess biological activity, e.g., incertain drug screening assays.

[0064] The expression systems that may be used for purposes of theinvention include, but are not limited to, microorganisms such asbacteria (e.g., E. coli, B. subtilis) transformed with recombinantbacteriophage DNA, plasmid DNA or cosmid DNA expression vectorscontaining NHK nucleotide sequences; yeast (e.g., Saccharomyces, Pichia)transformed with recombinant yeast expression vectors containing NHKnucleotide sequences; insect cell systems infected with recombinantvirus expression vectors (e.g., baculovirus) containing NHK nucleotidesequences; plant cell systems infected with recombinant virus expressionvectors (e.g., cauliflower mosaic virus, CaMV; tobacco mosaic virus,TMV) or transformed with recombinant plasmid expression vectors (e.g.,Ti plasmid) containing NHK nucleotide sequences; or mammalian cellsystems (e.g., COS, CHO, BHK, 293, 3T3) harboring recombinant expressionconstructs containing NHK nucleotide sequences and promoters derivedfrom the genome of mammalian cells (e.g., metallothionein promoter) orfrom mammalian viruses (e.g., the adenovirus late promoter; the vacciniavirus 7.5K promoter).

[0065] In bacterial systems, a number of expression vectors may beadvantageously selected depending upon the use intended for the NHKproduct being expressed. For example, when a large quantity of such aprotein is to be produced for the generation of pharmaceuticalcompositions of or containing NHK, or for raising antibodies to a NHK,vectors that direct the expression of high levels of fusion proteinproducts that are readily purified may be desirable. Such vectorsinclude, but are not limited, to the E. coli expression vector pUR278(Ruther et al., 1983, EMBO J. 2:1791), in which a NHK coding sequencemay be ligated individually into the vector in frame with the lacZcoding region so that a fusion protein is produced; pIN vectors (Inouye& Inouye, 1985, Nucleic Acids Res. 13:3101-3109; Van Heeke & Schuster,1989, J. Biol. Chem. 264:5503-5509); and the like. pGEX vectors may alsobe used to express foreign polypeptides as fusion proteins withglutathione S-transferase (GST). In general, such fusion proteins aresoluble and can easily be purified from lysed cells by adsorption toglutathione-agarose beads followed by elution in the presence of freeglutathione. The PGEX vectors are designed to include thrombin or factorXa protease cleavage sites so that the cloned target expression productcan be released from the GST moiety.

[0066] In an insect system, Autographa californica nuclear polyhedrosisvirus (ACNPV) is used as a vector to express foreign polynucleotidesequences. The virus grows in Spodoptera frugiperda cells. A NHK codingsequence can be cloned individually into non-essential regions (forexample the polyhedrin gene) of the virus and placed under control of anAcNPV promoter (for example the polyhedrin promoter). Successfulinsertion of NHK coding sequence will result in inactivation of thepolyhedrin gene and production of non-occluded recombinant virus (i.e.,virus lacking the proteinaceous coat coded for by the polyhedrin gene).These recombinant viruses are then used to infect Spodoptera frugiperdacells in which the inserted sequence is expressed (e.g., see Smith etal., 1983, J. Virol. 46: 584; Smith, U.S. Pat. No. 4,215,051).

[0067] In mammalian host cells, a number of viral-based expressionsystems may be utilized. In cases where an adenovirus is used as anexpression vector, the NHK nucleotide sequence of interest may beligated to an adenovirus transcription/translation control complex,e.g., the late promoter and tripartite leader sequence. This chimericsequence may then be inserted in the adenovirus genome by in vitro or invivo recombination. Insertion in a non-essential region of the viralgenome (e.g., region E1 or E3) will result in a recombinant virus thatis viable and capable of expressing a NHK product in infected hosts(e.g., See Logan & Shenk, 1984, Proc. Natl. Acad. Sci. USA81:3655-3659). Specific initiation signals may also be required forefficient translation of inserted NHK nucleotide sequences. Thesesignals include the ATG initiation codon and adjacent sequences. Incases where an entire NHK gene or cDNA, including its own initiationcodon and adjacent sequences, is inserted into the appropriateexpression vector, no additional translational control signals may beneeded. However, in cases where only a portion of a NHK coding sequenceis inserted, exogenous translational control signals; including,perhaps, the ATG initiation codon, must be provided. Furthermore, theinitiation codon must be in phase with the reading frame of the desiredcoding sequence to ensure translation of the entire insert. Theseexogenous translational control signals and initiation codons can be ofa variety of origins, both natural and synthetic. The efficiency ofexpression may be enhanced by the inclusion of appropriate transcriptionenhancer elements, transcription terminators, etc. (See Bitter et al.,1987, Methods in Enzymol. 153:516-544).

[0068] In addition, a host cell strain may be chosen that modulates theexpression of the inserted sequences, or modifies and processes theexpression product in the specific fashion desired. Such modifications(e.g., glycosylation) and processing (e.g., cleavage) of proteinproducts may be important for the function of the protein. Differenthost cells have characteristic and specific mechanisms for thepost-translational processing and modification of proteins andexpression products. Appropriate cell lines or host systems can bechosen to ensure the correct modification and processing of the foreignprotein expressed. To this end, eukaryotic host cells which possess thecellular machinery for proper processing of the primary transcript,glycosylation, and phosphorylation of the expression product may beused., Such mammalian host cells include, but are not limited to, CHO,VERO, BHK, HeLa, COS, MDCK, 293, 3T3, WI38, and in particular, humancell lines.

[0069] For long-term, high-yield production of recombinant proteins,stable expression is preferred. For example, cell lines that stablyexpress the NHK sequences described above can be engineered. Rather thanusing expression vectors which contain viral origins of replication,host cells can be transformed with DNA controlled by appropriateexpression control elements (e.g., promoter, enhancer sequences,transcription terminators, polyadenylation sites, etc.), and aselectable marker. Following the introduction of the foreign. DNA,engineered cells may be allowed to grow for 1-2 days in an enrichedmedia, and then are switched to a selective media. The selectable markerin the recombinant plasmid confers resistance to the selection andallows cells to stably integrate the plasmid into their chromosomes andgrow to form foci which in turn can be cloned and expanded into celllines. This method may advantageously be used to engineer cell lineswhich express the NHK product. Such engineered cell lines may beparticularly useful in screening and evaluation of compounds that affectthe endogenous activity of the NHK product.

[0070] A number of selection systems can be used including, but notlimited to, the herpes simplex virus thymidine kinase (Wigler, et al.,1977, Cell 11:223), hypoxanthine-guanine phosphoribosyltransferase(Szybalska & Szybalski, 1962, Proc. Natl. Acad. Sci. USA 48:2026), andadenine phosphoribosyltransferase (Lowy, et al., 1980, Cell 22:817)genes can be employed in tk⁻, hgprt⁻ or aprt⁻ cells, respectively. Also,antimetabolite resistance can be used as the basis of selection for thefollowing genes: dhfr, which confers resistance to methotrexate (Wigler,et al., 1980, Proc. Natl. Acad. Sci. USA 77:3567; O'Hare, et al., 1981,Proc. Natl. Acad. Sci. USA 78:1527); gpt, which confers resistance tomycophenolic acid (Mulligan & Berg, 1981, Proc. Natl. Acad. Sci. USA78:2072); neo, which confers resistance to the aminoglycoside G-418(Colberre-Garapin, et al., 1981, J. Mol. Biol. 150:1); and hygro, whichconfers resistance to hygromycin (Santerre, et al., 1984, Gene 30:147).

[0071] Alternatively, any fusion protein can be readily purified byutilizing an antibody specific for the fusion protein being expressed.For example, a system described by Janknecht et al. allows for the readypurification of non-denatured fusion proteins expressed in human celllines (Janknecht, et al., 1991, Proc. Natl. Acad. Sci. USA88:8972-8976). In this system, the sequence of interest is subclonedinto a vaccinia recombination plasmid such that the sequence's openreading frame is translationally fused to an amino-terminal tagconsisting of six histidine residues. Extracts from cells infected withrecombinant vaccinia virus are loaded onto Ni^(2+,) nitriloaceticacid-agarose columns and histidine-tagged proteins are selectivelyeluted with imidazole-containing buffers.

[0072] Also encompassed by the present invention are fusion proteinsthat direct the NHK to a target organ and/or facilitate transport acrossthe membrane into the cytosol. Conjugation of a NHK product to antibodymolecules or their Fab fragments could be used to target cells bearing aparticular epitope. Attaching the appropriate signal sequence to the NHKwould also transport the NHK to the desired location within the cell.Alternatively targeting of NHK or its nucleic acid sequence might beachieved using liposome or lipid complex based delivery systems. Suchtechnologies are described in “Liposomes:A Practical Approach”, New,R.R.C., ed., Oxford University Press, New York and in U.S. Pat. Nos.4,594,595, 5,459,127, 5,948,767 and 6,110,490 and their respectivedisclosures which are herein incorporated by reference in theirentirety. Additionally embodied are novel protein constructs engineeredin such a way that they facilitate transport of the NHK to the targetsite or desired organ, where they cross the cell membrane and/or thenucleus where the NHK can exert its functional activity. This goal maybe achieved by coupling of the NHK to a cytokine or other ligand thatprovides targeting specificity, and/or to a protein transducing domain(see generally U.S. applications Ser. No. 60/111,701 and 60/056,713,both of which are herein incorporated by reference, for examples of suchtransducing sequences) to facilitate passage across cellular membranesand can optionally be engineered to include nuclear localization.

5.3 Antibodies to NHK Products

[0073] Antibodies that specifically recognize one or more epitopes of aNHK, or epitopes of conserved variants of a NHK, or peptide fragments ofa NHK are also encompassed by the invention. Such antibodies include,but are not limited to, polyclonal antibodies, monoclonal antibodies(mAbs), humanized or chimeric antibodies, single chain antibodies, Fabfragments, F(ab′)₂ fragments, fragments produced by a Fab expressionlibrary, anti-idiotypic (anti-Id) antibodies, and epitope-bindingfragments of any of the above.

[0074] The antibodies of the invention can be used, for example, in thedetection of NHK in a biological sample and may, therefore, be utilizedas part of a diagnostic or prognostic technique whereby patients may betested for abnormal amounts of NHK. Such antibodies may also be utilizedin conjunction with, for example, compound screening schemes for theevaluation of the effect of test compounds on expression and/or activityof a NHK expression product. Additionally, such antibodies can be used nconjunction gene therapy to, for example, evaluate the normal and/orengineered NHK-expressing cells prior to their introduction into thepatient. Such antibodies may additionally be used as a method for theinhibition of abnormal NHK activity. Thus, such antibodies may,therefore, be utilized as part of treatment methods.

[0075] For the production of antibodies, various host animals may beimmunized by injection with the NHK, a NHK peptide (e.g., onecorresponding to a functional domain of a NHK), truncated NHKpolypeptides (NHK in which one or more domains have been deleted),functional equivalents of the NHK or mutated variant of the NHK. Suchhost animals may include, but are not limited to, pigs, rabbits, mice,goats, and rats, to name but a few. Various adjuvants may be used toincrease the immunological response, depending on the host speciesincluding, but not limited to, Freund's adjuvant (complete andincomplete), mineral salts such as aluminum hydroxide or aluminumphosphate, chitosan, surface active substances such as lysolecithin,pluronic polyols, polyanions, peptides, oil emulsions, and potentiallyuseful human adjuvants such as BCG (bacille Calmette-Guerin) andCorynebacterium parvum. Alternatively, the immune response could beenhanced by combination and or coupling with molecules such as keyholelimpet hemocyanin, tetanus toxoid, diphtheria toxoid, ovalbumin, choleratoxin or fragments thereof. Polyclonal antibodies are heterogeneouspopulations of antibody molecules derived from the sera of the immunizedanimals.

[0076] Monoclonal antibodies, which are homogeneous populations ofantibodies to a particular antigen, can be obtained by any techniquewhich provides for the production of antibody molecules by continuouscell lines in culture. These include, but are not limited to, thehybridoma technique of Kohler and Milstein, (1975, Nature 256:495-497;and U.S. Pat. No. 4,376,110), the human B-cell hybridoma technique(Kosbor et al., 1983, Immunology Today 4:72; Cole et al., 1983, Proc.Natl. Acad. Sci. USA 80:2026-2030), and the EBV-hybridoma technique(Cole et al., 1985, Monoclonal Antibodies And Cancer Therapy, Alan R.Liss, Inc., pp. 77-96). Such antibodies may be of any immunoglobulinclass including IgG, IgM, IgE, IgA, IgD and any subclass thereof. Thehybridoma producing the mAb of this invention may be cultivated in vitroor in vivo. Production of high titers of mAbs in vivo makes this thepresently preferred method of production.

[0077] In addition, techniques developed for the production of “chimericantibodies” (Morrison et al., 1984, Proc. Natl. Acad. Sci. USA81:6851-6855; Neuberger et al., 1984, Nature, 312:604-608; Takeda etal., 1985, Nature, 314:452-454) by splicing the genes from a mouseantibody molecule of appropriate antigen specificity together with genesfrom a human antibody molecule of appropriate biological activity can beused. A chimeric antibody is a molecule in which different portions arederived from different animal species, such as those having a variableregion derived from a murine mAb and a human immunoglobulin constantregion Such technologies are described in U.S. Pat. Nos. 6,075,181 and5,877,397 and their respective disclosures which are herein incorporatedby reference in their entirety. Also encompassed by the presentinvention is the use of fully humanized monoclonal antibodies asdescribed in U.S. Pat. No. 6,150,584 and respective disclosures whichare herein incorporated by reference in their entirety.

[0078] Alternatively, techniques described for the production of singlechain antibodies (U.S. Pat. No. 4,946,778; Bird, 1988, Science242:423-426; Huston et al., 1988, Proc. Natl. Acad. Sci. USA85:5879-5883; and Ward et al., 1989, Nature 341:544-546) can be adaptedto produce single chain antibodies against NHK expression products.Single chain antibodies are formed by linking the heavy and light chainfragments of the Fv region via an amino acid bridge, resulting in asingle chain polypeptide.

[0079] Antibody fragments which recognize specific epitopes may begenerated by known techniques. For example, such fragments include, butare not limited to, the F(ab′)₂ fragments which can be produced bypepsin digestion of the antibody molecule and the Fab fragments whichcan be generated by reducing the disulfide bridges of the F(ab′)₂fragments. Alternatively, Fab expression libraries may be constructed(Huse et al., 1989, Science, 246:1275-1281) to allow rapid and easyidentification of monoclonal Fab fragments with the desired specificity.

[0080] Antibodies to a NHK can, in turn, be utilized to generateanti-idiotype antibodies that “mimic” a given NHK, using techniqueswell-known to those skilled in the art. (See, e.g., Greenspan & Bona,1993, FASEB J. 7(5):437-444; and Nissinoff, 1991, J. Immunol.147(8):2429-2438). For example antibodies which bind to a NHK domain andcompetitively inhibit the binding of NHK to its cognate receptor/ligandcan be used to generate anti-idiotypes that “mimic” the NHK and,therefore, bind, activate, or neutralize a NHK, NHK receptor, or NHKligand. Such anti-idiotypic antibodies or Fab fragments of suchanti-idiotypes can be used in therapeutic regimens involving aNHK-mediated pathway.

[0081] Additionally given the high degree of relatedness of mammalianNHKs, the presently described knock-out mice (having never seen NHK, andthus never been tolerized to NHK) have a unique utility, as they can beadvantageously applied to the generation of antibodies against thedisclosed mammalian NHK (i.e., NHK will be immunogenic in NHK knock-outanimals).

[0082] The present invention is not to be limited in scope by thespecific embodiments described herein, which are intended as singleillustrations of individual aspects of the invention, and functionallyequivalent methods and components are within the scope of the invention.Indeed, various modifications of the invention, in addition to thoseshown and described herein will become apparent to those skilled in theart from the foregoing description. Such modifications are intended tofall within the scope of the appended claims. All cited publications,patents, and patent applications are herein incorporated by reference intheir entirety.

1 2 1 1368 DNA Homo sapiens 1 atgaggattg tttgtttagt gaaaaaccaacagcccctgg gagccaccat caagcgccac 60 gagatgacag gggacatctt ggtggccaggatcatccacg gtgggctggc ggagagaagt 120 gggttgctat atgctggaga caaactggtagaagtgaatg gagtttcagt tgagggactg 180 gaccctgaac aagtgatcca tattctggccatgtctcgag gcacaatcat gttcaaggtg 240 gttccagtct ctgaccctcc tgtgaatagccagcagatgg tgtacgtccg tgccatgact 300 gagtactggc cccaggagga tcccgacatcccctgcatgg acgctggatt gcctttccag 360 aagggggaca tcctccagat tgtggaccagaatgatgccc tctggtggca ggcccgaaaa 420 atctcagacc ctgctacctg cgctgggcttgtcccttcta accaccttct gaagaggaag 480 caacgggaat tctggtggtc tcagccgtaccagcctcaca cctgcctcaa gtcaacccta 540 tacaaggagg agtttgttgg ctacggtcagaagttcttta tagctggctt ccgccgcagc 600 atgcgccttt gtcgcaggaa gtctcacctcagcccgctgc atgccagtgt gtgctgcacc 660 ggcagctgct acagtgcagt gggtgccccttacgaggagg tggtgaggta ccagcgacgc 720 ccttcagaca agtaccgcct catagtgctcatgggaccct ctggtgttgg agtaaatgag 780 ctcagaagac aacttattga atttaatcccagccattttc aaagtgctgt gccacacact 840 actcgtacta aaaagagtta cgaaatgaatgggcgtgagt atcactatgt gtccaaggaa 900 acatttgaaa acctcatata tagtcacaggatgctggagt atggtgagta caaaggccac 960 ctgtatggca ctagtgtgga tgctgttcaaacagtccttg tcgaaggaaa gatctgtgtc 1020 atggacctag agcctcagga tattcaaggggttcgaaccc atgaactgaa gccctatgtc 1080 atatttataa agccatcgaa tatgaggtgtatgaaacaat ctcggaaaaa tgccaaggtt 1140 attactgact actatgtgga catgaagttcaaggatgaag acctacaaga gatggaaaat 1200 ttagcccaaa gaatggaaac tcagtttggccaattttttg atcatgtgat tgtgaatgac 1260 agcttgcacg atgcatgtgc ccagttgttgtctgccatac agaaggctca ggaggagcct 1320 cagtgggtac cagcaacatg gatttcctcagatactgagt ctcaatga 1368 2 455 PRT Homo sapiens 2 Met Arg Ile Val CysLeu Val Lys Asn Gln Gln Pro Leu Gly Ala Thr 1 5 10 15 Ile Lys Arg HisGlu Met Thr Gly Asp Ile Leu Val Ala Arg Ile Ile 20 25 30 His Gly Gly LeuAla Glu Arg Ser Gly Leu Leu Tyr Ala Gly Asp Lys 35 40 45 Leu Val Glu ValAsn Gly Val Ser Val Glu Gly Leu Asp Pro Glu Gln 50 55 60 Val Ile His IleLeu Ala Met Ser Arg Gly Thr Ile Met Phe Lys Val 65 70 75 80 Val Pro ValSer Asp Pro Pro Val Asn Ser Gln Gln Met Val Tyr Val 85 90 95 Arg Ala MetThr Glu Tyr Trp Pro Gln Glu Asp Pro Asp Ile Pro Cys 100 105 110 Met AspAla Gly Leu Pro Phe Gln Lys Gly Asp Ile Leu Gln Ile Val 115 120 125 AspGln Asn Asp Ala Leu Trp Trp Gln Ala Arg Lys Ile Ser Asp Pro 130 135 140Ala Thr Cys Ala Gly Leu Val Pro Ser Asn His Leu Leu Lys Arg Lys 145 150155 160 Gln Arg Glu Phe Trp Trp Ser Gln Pro Tyr Gln Pro His Thr Cys Leu165 170 175 Lys Ser Thr Leu Tyr Lys Glu Glu Phe Val Gly Tyr Gly Gln LysPhe 180 185 190 Phe Ile Ala Gly Phe Arg Arg Ser Met Arg Leu Cys Arg ArgLys Ser 195 200 205 His Leu Ser Pro Leu His Ala Ser Val Cys Cys Thr GlySer Cys Tyr 210 215 220 Ser Ala Val Gly Ala Pro Tyr Glu Glu Val Val ArgTyr Gln Arg Arg 225 230 235 240 Pro Ser Asp Lys Tyr Arg Leu Ile Val LeuMet Gly Pro Ser Gly Val 245 250 255 Gly Val Asn Glu Leu Arg Arg Gln LeuIle Glu Phe Asn Pro Ser His 260 265 270 Phe Gln Ser Ala Val Pro His ThrThr Arg Thr Lys Lys Ser Tyr Glu 275 280 285 Met Asn Gly Arg Glu Tyr HisTyr Val Ser Lys Glu Thr Phe Glu Asn 290 295 300 Leu Ile Tyr Ser His ArgMet Leu Glu Tyr Gly Glu Tyr Lys Gly His 305 310 315 320 Leu Tyr Gly ThrSer Val Asp Ala Val Gln Thr Val Leu Val Glu Gly 325 330 335 Lys Ile CysVal Met Asp Leu Glu Pro Gln Asp Ile Gln Gly Val Arg 340 345 350 Thr HisGlu Leu Lys Pro Tyr Val Ile Phe Ile Lys Pro Ser Asn Met 355 360 365 ArgCys Met Lys Gln Ser Arg Lys Asn Ala Lys Val Ile Thr Asp Tyr 370 375 380Tyr Val Asp Met Lys Phe Lys Asp Glu Asp Leu Gln Glu Met Glu Asn 385 390395 400 Leu Ala Gln Arg Met Glu Thr Gln Phe Gly Gln Phe Phe Asp His Val405 410 415 Ile Val Asn Asp Ser Leu His Asp Ala Cys Ala Gln Leu Leu SerAla 420 425 430 Ile Gln Lys Ala Gln Glu Glu Pro Gln Trp Val Pro Ala ThrTrp Ile 435 440 445 Ser Ser Asp Thr Glu Ser Gln 450 455

What is claimed is:
 1. An isolated nucleic acid molecule comprising anucleotide sequence that is described in SEQ ID NO:1.
 2. An isolatednucleic acid molecule comprising a nucleotide sequence encoding theamino acid sequence shown in SEQ ID NO:2.
 3. An isolated nucleic acidmolecule comprising a nucleotide sequence that: (a) encodes the aminoacid sequence shown in SEQ ID NO:2; and (b) hybridizes under stringentconditions to the nucleotide sequence of SEQ ID NO:1 or the complementthereof.